Functional Toll-like receptor 4 expressed in lactotrophs mediates LPS-induced proliferation in experimental pituitary hyperplasia

Toll like receptor 4 (TLR4) has been characterized for its ability to recognize bacterial endotoxin lipopolysaccharide (LPS). Considering that infections or inflammatory processes might contribute to the progression of pituitary tumors, we analyzed the TLR4 functional role by evaluating the LPS effect on lactotroph proliferation in primary cultures from experimental pituitary tumors, and examined the involvement of PI3K-Akt and NF-κB activation in this effect. In addition, the role of 17β-estradiol as a possible modulator of LPS-induced PRL cell proliferation was further investigated. In estrogen-induced hyperplasic pituitaries, LPS triggered lactotroph cell proliferation. However, endotoxin failed to increase the number of lactotrophs taking up BrdU in normal pituitaries. Moreover, incubation with anti-TLR4 antibody significantly reduced LPS-induced lactotroph proliferation, suggesting a functional role of this receptor. As a sign of TLR4 activation, an LPS challenge increased IL-6 release in normal and tumoral cells. By flow cytometry, TLR4 baseline expression was revealed at the plasma membrane of tumoral lactotrophs, without changes noted in the percentage of double PRL/TLR4 positive cells after LPS stimulus. Increases in TLR4 intracellular expression were detected as well as rises in CD14, p-Akt and NF-κB after an LPS challenge, as assessed by western blotting. The TLR4/PRL and PRL/NF-κB co-localization was also corroborated by immunofluorescence and the involvement of PI3K/Akt signaling in lactotroph proliferation and IL-6 release was revealed through the PI3K inhibitor Ly-294002. In addition, 17β-estradiol attenuated the LPS-evoked increase in tumoral lactotroph proliferation and IL-6 release. Collectively these results demonstrate the presence of functional TLR4 in lactotrophs from estrogen-induced hyperplasic pituitaries, which responded to the proliferative stimulation and IL-6 release induced by LPS through TLR4/CD14, with a contribution of the PI3K-Akt and NF-κB signaling pathways.     

White rot Basidiomycetes isolated from Chiloé National Park in Los Lagos region, Chile

Wood decomposition is an important component in forest ecosystems but information about the diversity of fungi causing decay is lacking. This is especially true for the temperate rain forests in Chile. These investigations show results of a biodiversity study of white-rot fungi in wood obtained from Chiloé National Park in Los Lagos region, Chile. Culturing from white-rotted wood followed by sequencing of the complete internal transcribed spacer region of the ribosomal DNA (rDNA) or partial large subunit region of the rDNA, identified 12 different species in the Basidiomycota. All of these fungi were characterized as white rot fungi and were identified with a BLAST match of 97 % or greater to sequences in the GenBank database. Fungi obtained were species of Phlebia, Mycoacia, Hyphodontia, Bjerkandera, Phanerochaete, Stereum, Trametes, and Ceriporiopsis. This report identifies for the first time in Chile the species Ceriporiopsis subvermispora, Hyphodontia radula, Phlebia radiata, Phanerochaete affinis, Peniophora cinerea, Stereum gausapatum, Phlebia setulosa and Phanerochaete sordida. Scanning electron microscopy was used to characterize the type of decay caused by the fungi that were isolated and a combination of selective lignin degraders and simultaneous white rot fungi were found. Fungi that cause a selective degradation of lignin are of interest for bioprocessing technologies that require modification or degradation of lignin without cellulose removal.     

Phosphorylation of C-terminal polycystin-2 influences the interaction with PIGEA14: A QCM study based on solid supported membranes

Polycystin-2 (PC2) trafficking has been proposed to be a result of the interaction of PIGEA14 with PC2 as a function of the phosphorylation state of PC2. Here, we investigated the interaction of PIGEA14 with the C-terminal part of polycystin-2 wild type (cPC2wt) and the pseudophosphorylated mutant (cPC2S812D) to first, quantify the binding affinity between cPC2 and PIGEA14 and second, to elucidate the influence of PC2 phosphorylation on PIGEA14 binding. Solid supported membranes composed of octanethiol/1,2-dioleoyl-sn-glycero-3-phosphocholine doped with the receptor lipid DOGS–NTA–Ni were used to attach PIGEA14 to the membrane via its hexahistidine tag. By means of the quartz crystal microbalance technique, binding affinities as well as kinetic constants of the interaction were extracted in a label-free manner by applying the scaled particle theory. The results show that the dissociation constant of cPC2 to PIGEA14 is in the 10 nM regime providing strong evidence of a very specific interaction of cPC2 with PIGEA14. The interaction of cPC2wt is twofold larger than that of cPC2S812D. The moderate higher binding affinity of cPC2wt to PIGEA14 is discussed in light of PC2 trafficking to the plasma membrane.     

Seasonal weather effects on hydrology drive the metabolism of non-forest lowland streams

Weather variations change stream hydrological conditions, affecting the stream function. A seasonal study in three well-conserved first-order Pampean streams was carried out to test the hypothesis that rainfalls are the main drivers of whole-stream metabolism, through their effects on hydrology. We estimated the stream metabolism and metabolic contribution of six relevant communities (angiosperms, macroalgae, seston, epiphyton, epipelon, and hyporheos) during late spring, summer, and winter and examined the relation between gross primary production (GPP) and photosynthetic active radiation (PAR). Our results showed that the decrease in available streambed light due to the dissolved organic carbon after rainfalls was the main factor related to the decrease in the ecosystem and community metabolisms. For instance, GPP oscillated from ~10 gO₂ m^?2 d^?1 in early spring (low flows) to ~3 gO₂ m^?2 d^?1 in summer (high flows). Ecosystem respiration (ER) was less sensitive than GPP to rainfalls due to the increase of hyporheic respiration. Rainfalls also caused a significant loss of downstream macroalgal biomass. At a day scale, the high PAR of late spring and summer saturated GPP during the afternoon, and the low temperature of winter mornings constrained GPP. Hence, the knowledge of weather changes is key to understanding the main hydrological drivers of stream function.     

In silico and in vitro studies to elucidate the role of Cu2+ and galanthamine as the limiting step in the amyloid beta (1–42) fibrillation process

The formation of fibrils and oligomers of amyloid beta (Aβ) with 42 amino acid residues (Aβ_1–42) is the most important pathophysiological event associated with Alzheimer''s disease (AD). The formation of Aβ fibrils and oligomers requires a conformational change from an α-helix to a β-sheet conformation, which is encouraged by the formation of a salt bridge between Asp 23 or Glu 22 and Lys 28. Recently, Cu²⁺ and various drugs used for AD treatment, such as galanthamine (Reminyl®), have been reported to inhibit the formation of Aβ fibrils. However, the mechanism of this inhibition remains unclear. Therefore, the aim of this work was to explore how Cu²⁺ and galanthamine prevent the formation of Aβ_1–42 fibrils using molecular dynamics (MD) simulations (20 ns) and in vitro studies using fluorescence and circular dichroism (CD) spectroscopies. The MD simulations revealed that Aβ_1–42 acquires a characteristic U-shape before the α-helix to β-sheet conformational change. The formation of a salt bridge between Asp 23 and Lys 28 was also observed beginning at 5 ns. However, the MD simulations of Aβ₁₋₄₂ in the presence of Cu²⁺ or galanthamine demonstrated that both ligands prevent the formation of the salt bridge by either binding to Glu 22 and Asp 23 (Cu²⁺) or to Lys 28 (galanthamine), which prevents Aβ₁₋₄₂ from adopting the U-characteristic conformation that allows the amino acids to transition to a β-sheet conformation. The docking results revealed that the conformation obtained by the MD simulation of a monomer from the 1Z0Q structure can form similar interactions to those obtained from the 2BGE structure in the oligomers. The in vitro studies demonstrated that Aβ remains in an unfolded conformation when Cu²⁺ and galanthamine are used. Then, ligands that bind Asp 23 or Glu 22 and Lys 28 could therefore be used to prevent β turn formation and, consequently, the formation of Aβ fibrils.     

Diversity and expression of P450 genes from Dendroctonus valens LeConte (Curculionidae: Scolytinae) in response to different kairomones

Bark beetles (Curculionidae: Scolytinae) are major cause of woody plants death in the world. They colonize the stem and other parts of trees recognizing host-produced specific compounds (kairomones) and insect pheromones. Bark beetle's antennae and alimentary canal participate in the host selection identifying chemical compounds produced by trees and insects, and also in the metabolism and detoxification of these compounds. The red turpentine beetle (RTB), Dendroctonus valens LeConte, is an unaggressive species that colonize > 40 pine species (Pinaceae) in North and Central America. Several studies suggest that bark beetle cytochrome P450 enzymes are involved in monoterpene oxidation. In this study we identified by means of PCR, cloning, sequencing, and bioinformatic analysis, eleven full-length genes: five CYP4, four CYP6, and two CYP9 in the antennae and gut region of RTB, after stimulation with vapors of monoterpenes: (±)-α-pinene, (R)-(+)-α-pinene, (S)-(?)-β-pinene, (S)-(?)-α-pinene and (+)-3-carene; pine trees volatiles used by RTB as kairomones. The recovered cDNA of these genes vary from 1.5 kb to 1.8 kb and the open frame encodes from 496 to 562 amino acid proteins. The bioinformatic analysis suggests that the majority of P450 proteins encoded by these genes are membrane anchored in the endoplasmic reticulum. RT-qPCR assays showed differential expression of all CYP genes between male and female. The gene expression was dependent of monoterpenes and exposure time, with some of them sex, antennae and gut region specific. Significant differences among monoterpenes, gut region, antennae and exposure time were found. Our results suggest that some of these genes may be involved in the detoxification process of these compounds during tree colonization.     

Synthesis and biological evaluation against Leishmania amazonensis of a series of alkyl-substituted benzophenones

Nine O-alkyl and O-prenyl derivatives were synthesized from commercial 2,4-dihydroxybenzophenone, 4,e4,4′-dihydroxybenzophenone and were evaluated for their leishmanicidal activity against promastigote forms of Leishmania amazonensis, as well their toxicity in murine macrophages. All derivatives exhibited better biological activity than their hydroxylated benzophenones precursors, and new compound LFQM-123 (3c) was 250-fold more active than its precursor 4,4′-dihydroxybenzophenone (3). Moreover, some of the results were comparable to the standard drug Amphotericin B, suggesting that the increase in lipophilicity could facilitate protozoa membrane permeation. In this study we confirmed that benzophenone derivatives exhibit leishmanicidal properties, with relatively low toxicity, and thus could be exploited as promise prototypes for the design and development of new drug for the treatment of leishmaniasis.     

Luteibacter rhizovicinus MIMR1 promotes root development in barley (Hordeum vulgare L.) under laboratory conditions

In order to preserve environmental quality, alternative strategies to chemical-intensive agriculture are strongly needed. In this study, we characterized in vitro the potential plant growth promoting (PGP) properties of a gamma-proteobacterium, named MIMR1, originally isolated from apple shoots in micropropagation. The analysis of the 16S rRNA gene sequence allowed the taxonomic identification of MIMR1 as Luteibacter rhizovicinus. The PGP properties of MIMR1 were compared to Pseudomonas chlororaphis subsp. aurantiaca DSM 19603^T, which was selected as a reference PGP bacterium. By means of in vitro experiments, we showed that L. rhizovicinus MIMR1 and P. chlororaphis DSM 19603^T have the ability to produce molecules able to chelate ferric ions and solubilize monocalcium phosphate. On the contrary, both strains were apparently unable to solubilize tricalcium phosphate. Furthermore, the ability to produce 3-indol acetic acid by MIMR1 was approximately three times higher than that of DSM 19603^T. By using fluorescent recombinants of strains MIMR1 and DSM 19603^T, we also demonstrated that both bacteria are able to abundantly proliferate and colonize the barley rhizosphere, preferentially localizing on root tips and in the rhizoplane. Finally, we observed a negative effect of DSM 19603^T on barley seed germination and plant growth, whereas MIMR1, compared to the control, determined a significant increase of the weight of aerial part (+22 %), and the weight and length of roots (+53 and +32 %, respectively). The results obtained in this work make L. rhizovicinus MIMR1 a good candidate for possible use in the formulation of bio-fertilizers.     

Chemical speciation and competitive cationic partitioning on a sandy aquifer material

Abstract

Coupled geochemical speciation/transport models are being developed to assess potential transport of metal contaminants in the subsurface environment. In a test of the geochemical speciation portion of the effort, MINTEQA2 model predictions are compared with laboratory data concerning the pH dependent partitioning behavior of eight cationic contaminants (Ba, Be, Cd, Cu, Ni, Pb, Tl and Zn) on a sandy aquifer material in an oxidized environment. MINTEQA2 contains provisions for describing potential attenuation due to both mineral phase precipitation processes and adsorption processes resulting from amorphous iron oxides in aquifer materials (MIT Diffuse Layer Model). In the comparison, several trends were discerned. (1) Adsorptive processes tend to better describe the pH-dependent partitioning behavior of transition metals (especially Pb, Zn and Ni). (2) Cd behavior is better described by precipitation as a cadmium carbonate phase. (3) Cu behavior is not reasonably described by the model. (4) Ba and Be comparisons are poor (although presumably their partitioning behavior results from adsorptive and/or pH sensitive solid solution processes). (5) unlike the other elements, the behavior of Tl is relatively insensitive to pH.     

Comparing galling insect richness among Neotropical savannas: effects of plant richness, vegetation structure and super-host presence

Plant species diversity maintains the stability of ecosystems and the diversity of consumer species such as insect herbivores. Considering that gall-inducing insects are highly specialized on their host plants and dependent on the occurrence, abundance and distribution of plants, we evaluated the diversity patterns of gall-inducing insect along Brazilian Neotropical savannas and the potential role of plant species richness, vegetation structure and super-host presence on determining these patterns. We found 1,882 individual plants that belonged to 64 different host plant species grouped in 31 families, associated to 112 galling insect species. The galling richness was positively influenced by plant species richness and the presence of the super-host genus Qualea (Vochysiaceae). Plant species richness explained 48 % of the galling richness and areas with presence of super-hosts had more than twice of galling species than areas where they were absent. On the other hand, we found no evidence that larger plants hosted more species of galling insects. We observed that for the diversity of galling insects in the Brazilian Cerrado, vegetation structure explained almost the same portion as plant richness, because structural variables did not have an effect on residuals of galling richness and plant richness regression. Our findings suggests that plant richness has a more important role on the mitigation of natural enemies and adaptive radiation of galling species, while structural aspects of the vegetation does not seem to have that effect. Furthermore, we show that the super-host taxa provide an increment in local galling richness because they present a great diversity of local number of gall morphospecies (i.e. alpha diversity) and the high turnover of morphospecies among different localities (i.e. beta diversity). Therefore we argue that the quality of resources (richness and super host presence) appears to be a most important factor for the diversity of galling insects in Neotropical systems, than the amount of resources.